Soybeans, wheat and barley contain .alpha.-amylase in addition to .beta.-amylase. In order to remove the .alpha.-amylase, a method of deactivating .alpha.-amylase by varying the ambient pH and temperature or by using a chelating agent has heretofore been used. However, since complete removal of .alpha.-amylase by the method is accompanied by deactivation of .beta.-amylase, it is difficult to completely remove .alpha.-amylase by the method in view of the yield of .beta.-amylase.
On the other hand, in the starch saccharification industry, production of a high-purity maltose syrup is being advanced and therefore a high-purity .beta.-amylase which does not produce any other saccharides to be derived from .alpha.-amylase than maltose is needed.
There is known affinity chromatography as a method of rapid and specific purification of enzyme proteins. In accordance with the method, only the intended enzyme is specifically adsorbed to an insoluble carrier having a substrate of the enzyme or a substrate analogue thereof as fixed thereto so that the soluble impurities are removed from the enzyme, whereby only the intended substance (enzyme) is specifically and rapidly separated and purified.
Some reports have already been disclosed, regarding affinity chromatography of .beta.-amylase. For instance, Per Vretblad (FEBS Letters, 47 (1974) 86-89) reported that .beta.-amylase from sweet potatoes is specifically adsorbed to a Sepharose column having .alpha.-cyclodextrin, which is a .beta.-amylase antagonistic inhibitor, as fixed thereto, whereupon a bovine serum albumin, which has been added to the column simultaneously as a model of an impurity protein, is not adsorbed to the column and may therefore be removed from .beta.-amylase, and that the adsorbed .beta.-amylase may specifically be eluted from the column by adding .alpha.-cyclodextrin to the eluent. A. Hoschke et al. (Starch, 28 (1976), 426-432) reported, though not explicitly referring to the origin, that .beta.-amylase adsorbs to a Sepharose column having .alpha.-cyclodextrin as fixed thereto.
However, recently, K. Subbaramiah et al. (Starch, 41, (1989) 357-359) disclosed a different report mentioning that when they prepared Sepharose having cyclodextrin as fixed thereto by the same method as the above mentioned Per Vretblad's method (FEBS Letters, 47, 1974), any .beta.-amylase derived from sweet potatoes, potatoes and malt did not adsorb it.
Under the situation, we the present inventors prepared and examined an .alpha.-cyclodextrin-fixed Sepharose column in accordance with the Per Vretblad's report (FEBS Letters, 47, 1974) and found that .beta.-amylase derived from soybeans and malt, which has an important role in the starch saccharification industry, does not adsorb to the carrier. At present, therefore, there is no report relating to specific affinity chromatography of .beta.-amylase derived from soybeans and malt.
Under the above-mentioned conditions, we the present inventors earnestly investigated affinity chromatography for adsorbing .beta.-amylase derived from soybeans and malt, which has an important role in the starch saccharification industry, and, as a result, have found that such .beta.-amylase which does not adsorb to the .alpha.-cyclodextrin-fixed carrier in the presence of a buffer or the like may efficiently adsorb to thereto by adding ammonium sulfate to the solution to be applied to the column. On the basis of the finding, we have completed the present invention.